1. Field of Invention
The present invention relates to the separation of hemicellulases from a mixture thereof. In particular the present invention relates to the separation of xylanase from a mixture thereof from other hemicellulases, particularly cellulase to produce a xylanase rich fraction which is subsequently purified and concentrated and is particularly useful in the hydrolysis of hemicelluloses for which it is selective and thus is useful for hydrolysing hemicelluloses in the presence of celluloses. Also produced is a concentrated cellulase rich fraction which is useful in the hydrolysis of cellulose.
2. Description of the Prior Art
Lignocellulose, the world's largest renewable biomass resource is composed mainly of lignin, cellulose and hemicellulose, of which the large part of the latter is xylan. Much of the current research and development has been directed towards the utilization of the cellulose fraction for liquid fuel production. However if value-added products could be obtained from the hemicellulose and lignin streams, economics of the process could be significantly improved. Applicants are presently developing a process for the production of fuel and chemicals from lignocellulosics. In the process lignocellulosics are first pretreated with steam then fractionated into hemicellulose and lignin streams. Both cellulose and hemicellulose are potential sources of sugar for fermentation into valuable products. Thus for a number of years the applicants have been developing a process where aspenwood chips are first steam pretreated to enhance their subsequent enzymatic hydrolysis to fermentable sugar. As disclosed in Saddler, et al (1983) Biotechnol. Bioeng. Symp. 13, 225-238, the hemicellulose, lignn, and cellulose streams were then separated by selectively extracting the pretreated material with water and dilute alkali. The cellulose and hemicellulose streams were then respectively hydrolysed by cellulases and xylanases and fermented to ethanol and 2,3-butanediol by the appropriate microorganisms.
It had further been shown that high levels of cellulase and xylanase enzymes were efficiently produced by the fungus Trichoderma harzianum E 58 as disclosed by Yu, et al (1984) Biotechnol. Bioeng. Symp. 14, 341-352. As the hemicellulose and cellulose derived sugars are normally utilized by different microorganisms for various products, the hemicellulose and cellulose fractions are routinely separated into different streams in the above process and thus if an inexpensive process is available for the separation of the xylanase complex from the cellulase complex two enzyme streams could be efficiently utilized for the hydrolysis of the hemicellulose and cellulose streams, respectively, while reducing the overall cost of separate enzyme production steps.
D-xylanases have been purified from many sources using various combinations of techniques such as ion-exchange chromatography, gel permeation chromatography, isoelectric focusing, zone electrophoresis, affinity binding and crystallization. Attention is directed to the article of Dekker, R. F. H. and Richards, G. N. (1976) Adv. Carbohydrl Chem. Biochem. 32, 277-352. However the described procedures are deficient in that they are either too slow, too cumbersome to use, too difficult to scale up or too expensive to incorporate into the production scheme for a bulk enzyme. Alternatively, others as disclosed by Paice, M. G. et al (1984), J. Wood Chem. Technol. 4, 187-198 have used ethanol to selectively separate the xylanase from the cellulase components but this required very high concentrations of ethanol and the purified xylanases still contained approximately 3% cellulase. In addition, the use of organic solvents necessitates the need for the wastewater treatment, and devices for the removal of toxic and explosive vapours as well as explosive proof motors and switches as disclosed by Volesky, B. et al (1985) CRC Crit. Rev. Biotechnol 2 119-146. These can all add to the capital and processing costs. Still others have prepared small amounts of cellulase-free xylanase by cloning the xylanase gene into non-cellulolytic microorganisms as disclosed in the poster session at the Pulp and Paper Research Institute of Canda, Pointe Claire, Quebec, 1985. The problems associated with this approach is that the xylanase enzyme synthesized by the new host is usually located intracellularly. The extraction of the xylanase enzyme from inside the cell is difficult, time-consuming, expensive and results in low yield. The xylanase, unless fractionated by a series of complicated procedures, will be of low specific activity and may contain proteases which may destabilize the xylanase activities. Thus processes for the purification of enzymes including xylanases are already available. However, these processes suffer from being cumbersome, requiring many steps which generally result in low recoveries. The processes are often time consuming with low yields of the product xylanase (milligram quantities). These processes are mainly designed for the preparation of small amounts of xylanase, irrespective of the cost in time and money for the purposes of analytical studies. Such processes are not feasible if the xylanase is to be used on an industrial scale. Purification processes are disclosed by Frederick, et al in an article entitled "Purification and Characterization of Endo-xylanases from Aspergillus niger" Biotechnology and Bioengineering, Vol. 27, pages 525-532 (1985). Again reference is made to an article entitled "Xylan-Degrading Enzymes of the Yeast Cryptococcus albidus" by Biely, et al in European Journal of Biochemistry, 108, pages 313-321 and an article of "Isolation and Characterization of a Xylanase from Bacillus subtilis, Vol. 46 No. 2, Applied Environmental Microbiology, Aug. 1983, pages 511-514, a further article entitled "Purification and Some Properties of an Endo 1,4-.beta.-D-xylanase from Streptomyces sp, by Tasuku Nakajima, et al in the Journal of Ferment. Technol. Vol. 62, No. 3, pages 269-276, 1984, an article entitled "Purification and Some properties of Xylanase from Cryptococcus flavus by Nakanishi, et al, Journal of Ferment. Technol. Vol. 62, No. 4, pages 361-369, 1984 and an article entitled Isolation, Purification and Some of the Properties of Hemicellulase from Fusarium Sp.by Wankhede, et al, Carbohydrate Research, 96 (1981) pages 249-257.